This project addresses specific safety-related problems which are unique to herpesvirus based biological products. 1) These viruses establish latency and reactivate, presenting unique safety concerns with respect to their administration as live vaccines (as occurs with live varicella vaccine). Using herpes simplex virus (HSV) as a model, molecular virology techniques are being used to identify the genes involved in latency and reactivation, and appropriate methods for attenuating these viruses are being studied. Mutant viruses unable to produce the HSV latency-associated transcripts (LATs) during latency were constructed and shown to be deficient in reactivation from latently infected guinea pigs and mice. Experiments usin clinical virus isolates demonstrated that the quantity of LAT does not influence reactivation patterns. Mutant viruses with altered kinetics of LAT transcription were constructed, and used to identify interactions with other HSV genes. Intertypic viral recombinants between HSV-1 and HSV-2 wer made and are being used to determine whether LAT influences the site specificity of viral reactivation in animal models. Other experiments focu on the regulation of the LATs. 2) Herpesviruses are capable of providing helper function to other viruses (e.g. adeno-associated virus) which are no detected by currently used cell substrate testing techniques. As no well standardized test is in place to test for potential contamination of vaccines, another goal of this project is to identify means by which vaccines can be tested for such agents. A polymerase chain reaction based assay for vaccines is being developed. 3) Herpesviruses may play a role as vectors in neuronal gene therapy or intracellular immunization. Techniques for attenuation of these vectors to eliminate potential rescue of infectiou virus or damage to cells are being studied. Attenuated HSV strains are bein further mutated, and their ability to deliver foreign genes and antisense RNAs to cells in tissue culture and animals will be tested.